title: Thymoquinone inhibits microtubule polymerization by tubulin binding and causes mitotic arrest following apoptosis in A549 cells.
abstract: Microtubule-Targeting agents (MTA) are indispensable for cancer therapeutics. We here report thymoquinone (TQ) as a new MTA that already has been appreciated for its anticancer effects. TQ induced G2/M cell cycle arrest in human non-small lung epithelial cells (A549) and majority of arrested cells were in mitosis. TQ depolymerized the microtubule (MT) network and disrupted mitotic spindle organization of A549 cells. MT depolymerization by TQ was followed by apoptosis and subsequent loss in cell viability (IC50 value of ∼10 μM). Interestingly, TQ didn't affect the MT network of normal HUVEC cells at and below the IC50 concentration for A549 cells. TQ also inhibited tubulin polymerization in cell-free system with an IC50 of 27 μM and bound to tubulin heterodimers at a single site with a dissociation constant of 1.19 μM at 25 °C. Binding of TQ to tubulin quenched the tryptophan fluorescence of protein in a time-dependent manner. The TQ-tubulin binding kinetics was biphasic in nature and equilibrated in 30 min. TQ competed with colchicine for tubulin binding with a Ki of 1.9 μM as determined by modified Dixon plot analysis, this suggests TQ may bind tubulin at or near the colchicine binding site and in silico modeling study supported that. Our results establish a novel antimitotic mechanism of TQ by its direct binding to tubulin-MT network in A549 cells.
authors.affiliation: Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India.
authors.forename: Bipul R
authors.initials: BR
authors.lastname: Acharya
authors.affiliation: Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India.
authors.forename: Abhisek
authors.initials: A
authors.lastname: Chatterjee
authors.affiliation: Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India.
authors.forename: Arnab
authors.initials: A
authors.lastname: Ganguli
authors.affiliation: Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India.
authors.forename: Surela
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India. Electronic address: gcbcg@caluniv.ac.in.
authors.forename: Gopal
authors.initials: G
authors.lastname: Chakrabarti
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D002474:Cell-Free System; D003078:Colchicine / Q000494:pharmacology; D054697:Cytostatic Agents / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects*; D008938:Mitosis / Q000187:drug effects*; D009928:Organ Specificity; D058105:Polymerization; D013050:Spectrometry, Fluorescence; D008941:Spindle Apparatus / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: D016227:Benzoquinones; D054697:Cytostatic Agents; D014404:Tubulin; D050257:Tubulin Modulators; C003466:thymoquinone; D003078:Colchicine
keywords: (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide); 1,4-piperazinediethanesulfonic acid; 4′,6-diamidino-2-phenylindole; Apoptosis; Cancer therapy; DAPI; DMSO; EGTA; GTP; MT polymerization inhibition; MTT; Mitotic arrest; PI; PIPES; ROS; TQ; Thymoquinone; dimethyl sulfoxide; ethylene bis(oxyethylenenitrilo) tetraacetic acid; guanosine 5′-triphosphate; propidium iodide; reactive oxygen species; thymoqionone